Removal of C-terminal peptide amides from a 3-nitro-4-aminomethyl-benzoyl amide resin by photolysis

100.5, pp JO1 - 304, 1975.

Tetmbodron Letton

Per&smotl Press. PrintedlaQwtBritaia.

RBMOVAL OF C-TERMINAL 3-NITRO-4-AMINOM%THYL-BENZOYL

PEPTIDE AMIDES FROM A AMIDE RESIN BY PHOTOLYSIS K.

Daniel H. Rich and S. School of Pharmacy,

University

Gurwara

of Wisconsin,

Madison, Wisconsin

53706

(Roceivsdin UBA 28 Ootober 1974; raoeived in UK for abliostion 23 Ibomber 1974) The C-terminal

amide group is present

including deamino-oxytocin, 1 LH_m;*'3

However,

these conditions

groups which restrict

are

as the valine methods

to amminolysis

Furthermore,

in secretin.

for preparing

C-terminal

amides

4-aminomethylbenzoylamide

can be difficult

Resin 1 was prepared with N-bromosuccinimide 81% yield7"

acid

derivative &by clohexylamine

to illustrate

4

such

Thus new

from a solid phase as the

Protected peptides

can be removed

under conditions which do not The syn-

the method. Bromination

of E-toluic

gave *brano-E-toluic

acid

acid

(L)

(2) in

of 2 in 90% nitric acid at -lO°C for 2.5 hr gave 3acid

(1) in 85% yield,8 which upon reaction

in 72% yield.

(mp 205-207OC)

by coupling the Boc-nitro

converted

to its Boc-

in D14S08 anti characterized

(85%).

in liquid

gave 3-nitro-4-aminomethylbenzoic

Amino acid &was

reaction with Boc-azide salt z

residues.

groups nor aranatic amino acids.

for 48 hr at 4OC in a pressure bottle

(2) (mp 235-237OC)

prepared

resin 1.

in refluxing benzene

nitro-4-brancmethylbenzoic ammonia

peptides

as shown in Scheme I.

Nitration

C-terminal

to remove from the resin.

amide by photolysis

dedtroy acid or base labile protecting thesis of LH-RH is presented

and, therefore,

We report here the synthesis of the 3-nitro-

polystyrene

from resin 1 as the C-terminal

6

groups which can be used to synthe-

peptides with hindered

be useful.

or transesterification.

or transesterification,

and removing protected

would

amide of the pro-

the use of side chain ester protecting

the type of acid labile protecting

size the peptide.

active peptides

These peptides have been

from the resin by amminolysis necessitate

resistent

and secretin.

4

in which the C-terminal

synthesized by solid phase methods' tected peptide was removed

in several biologically

Resin 1

as tbe dicy-

(0.23 mmol/g Boc-NH) WaS

acid 5a in DMF using DCC with the aminomethyl

302

MO. 5

(0.23 mmol/g NH2), which was prepared by amination

resin 5

polystyrene chloride.'**

of chloromethylated

(0.23 mmol/g Cl, 1% divinyl benzene) with liquid ammonia

in methylene

Resin 2 was prepared by treating resin 2 with 50% TFA/CH2C12

followed by neutralization

with triethylamine

prepared by coupling Boc-Gly

in CH2C12.

Resins 2 and J& were

and Boc-Val with resin 2 in DMF using DCC.

Scheme I BocNR CH2Br $3

I

$2B=

CO,H

C02E

CO,H

5a (=2*2 3

Resin 0 u-

y2m2

C02R

P2

C02R

Re~iie&l~Ni-@CR:-R

cRC13

NO2 1,

a

R -

Boc

t33R=I.l

R - Boc-Glyl0? B - Boc-Val2,

Protected (Scheme II).

amino acid amides were formed upon photolysis The resin was suspended

absence of oxygen. 3200 3.

and Boc-Val-NH2

that hindered

at 3500 4 in the

(11)' were isolated in quantitative

amino acid derivatives

from resin -10 under mild conditions. group

and irradiated

A 40% CuSO4 solution was used to filter out wavelengths

Boc-Gly-NH2

which established

in methanol

of resins 2 and 10

can be removed

below

yield,

efficiently

In addition to the Boc group, the 0-benzyl

(on Ser, Tyr, Asp and Glu), and the tosyl group

(on Arg and His) are not

removed by photolysis. Scheme II

The C-terminal III).

Boc-amino

procedure

peptide

using resin 2

(Scheme

acids were coupled stepwise with resin 2 with DCC following the

of Merrifield.4

as described

amide, LH-RH -14 was synthesized

The protected

and after chromatography

decapeptide

resin

and crystallization

(2)

was photolyzed

the protected

decapep-

303

No. 5

tide amide -13 was isolated 8 Gly-resin 2. Scheme

in analytically

pure form in 65% yield based on Boc-

III

O-R synthesis

Na/NE$(liq) p-Glu-His-Trp-Ser-Tyr-Gly-I,eu-Arg-Pro-Gly-RR2 14 -

Decapeptide

13 was converted 10

to LPI-RR -14 by reaction with sodium in liquid

The LPI-RR (l4) was isolated

ammonia

aa described.

parable

to the yields we obtain

in 46% yield

(which is com-

for this reaction on LH-RH prepared

dard solid phase resins) and shown to be identical with authentic protected prepared

heptapeptide

LH-RR. *

The

Boc-Ser(Bzl)-'Pyr(Bzl)-Gly-Leu-Arg(Tos)-Pro-Gly-NEI2

by this method was isolated

Boc-Gly-resin

using stan-

in 56% yield

also

(182 mg),8 based on starting

2, and shown to be identical with a sample prepared by standard

solid phase synthesis. These results establish peptides

as the C-terminal

C-terminal conditions. amide which

that resin 1 can be used to synthesize amides in good yield on a preparative

protected

scale.

Hindered

amino acids, e.g. Val, Leu, Phe, are removed under normal photolysis Thus resin 1 can be useful is difficult

to remove

for the synthesis

from the Merrifield

of C-terminal

peptide

resin under mild condi-

tions. This work was supported by a grant from the National (AM-15507). partial

We thank the Graduate

Institutes

School of the University

of Health

of Wisconsin

for

support of this work.

References 1.

H. Takashima, 1323

(1968).

V. du Vigneaud,

and R. B. Merrifield,

J. Amer. Cbem. &.,

90,

2.

A. V. Schally, A. Arimura",Y. Baba, R. M. G. Nair, Ii.Matsuo, T. W. Redding, L. Debeljuk, and W. F. White, Biochem. Bionhvs. m.

3.

Abbreviations used are:

m.,

43, 393 (1971).

LH-RH, luteinizing hormone-releasing hormone: TFA,

trifluoroacetic acid; DCC, dicyclohexylcarbodiimide; Tos, tosyl; BOC, wbutoxy carbonyl; TEA, triethylamine. 4.

M. Bodansky and J. T. Sheehan, -Chem. Ind. (London), 1597 (1966).

5.

R. B. Merrifield, J. Amer. Chem. a.,

85, 2149 (1963); Intra-Sci. Chem.

Reports, 3, 183 (1971). 6.

El.C. Beyennan, H. Hindriks, and E. W. B. de Leer, 2. C. S. Chem. Comm., 1668 (1968).

7.

D. H. Rich and S. K. Gurwara, 168th National Meeting of the American Chemical Society, Atlantic City, N. J., Sept. 1974, Orgn. 125.

8.

Satisfactory microanalysis, amino acid analysis, mar, ir, tic, and mass spectral data were obtained.

9.

R. Schwyzer, A. Costopanagiotin, and P. Sieber, -Helv. Chim. m,

‘$5, 870

(1970). .O.

M. Manning, E. Coy, and A. Schally in Chemistry and Biology of Peptides, J. Meienhofer, ed., Ann Arbor Science Publishers, 1972, pp. 235-238.